| Background:Although the cornea is a physiologically transparent tissue, pathologic corneal neovascularization (CNV) is observed after inflammation caused by infection, aberrant immune responses, or chemical or thermal burns. CNV is a sight-threatening condition which is characterized by the vessels growing into the transparent cornea from corneal limbus. The balance between angiogenic and antiangiogenic factors determines the fate of CNV during wound healing. However, the mechanisms involved in the induction of CNV are complex and treatments of CNV with drugs, lasers, and surgery have demonstrated variable and largely limited clinical success. Therefore, it is worthwhile to explore a method to treat with low cost and less side effects for clinical application.Retinoic acid (RA), a vitamin A derivative, is a potent regulator of cell growth, differentiation, and matrix formation in various types of cells. RA is currently used to diseases such as psoriasis and acne. Previous studies have demonstrated that RA can inhibit the proliferation of RPE cells by disrupting some integrin functions and has low cytotoxic effects. Recent studies have demonstrated that RA in vivo suppresses inflammatory responses and tissue damage, and ameliorates inflammation in animal models of autoimmune diseases.It is widely known that alkali burns in the corneal cause abundant CNV in which severe inflammation and corneal lysis are involved, so alkali burns in animals are often used as a neovascularization model.Objectives:This study aims to investigate the effects of retinoic acid on experimental corneal neovascularization in alkali-burn induced SD rats and to explore its mechanism by using human dermal microvascular endothelial cell (HDMEC) in vitro.Materials and methods:CNV was induced in vivo by alkali burns on the corneas of SD rat. A total of 120 rats were randomly divided into 4 groups (n=30). Group 1:Control group, without alkali burn; Group 2:alkali burn with PBS treatment; Group 3:alkali burn with retinoic acid treatment and group 4:alkali burn with co-treatment with retinoic acid and thrombospondin-1 blocking peptide (TBP) treatment. The areas of CNV were measured on a daily basis under stereoscopic microscope in a blinded fashion on day 2,5,10 after alkali burns. The protein levels of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) in corneas were measured by Western blotting on days 2,5 and 10. HDMEC cell proliferation, migration and differentiation assay were performed after treated with retinoic acid in vitro. Amount of TSP-1 secreted from HDMEC in the culture medium were measured by ELISA. TSP-1 gene expression in HDMEC was measured by qPCR and protein level was confirmed by Western blotting.Results:TSP-1 expression decreased and VEGF expression increased after alkali burn in the corneal. Corneal TSP-1 protein levels measured by Western blotting were higher in the RA-treated group, but VEGF protein levels were lower in the RA-treated group in time-dependent manner compare to the PBS-treated group after alkali burns (p<0.05). Sprouting of new vessels were decreased in the RA-treated group compare to the PBS-treated group after 10 days of alkali burn, however, this effect was attenuated in the RA with thrombospondin-1 blocking peptide (TBP) co-treated group. RA increases TSP-1 expression in HDMEC but not affect release of TSP-1 from HDMEC in culture medium. In addition, RA inhibits proliferation of HDMEC and induces apoptosis of HDMEC. Moreover, RA inhibits tube formation of HDMEC, but this effect attenuated by TBP treatment. However, RA showed no effect on migration of HDMEC.Conclusions:RA treatment reduced alkali-burn induced corneal neovascularizaiton in SD rats. RA protected the cornea from chemical damage via over production of TSP-1 and decreased expression of VEGF in vivo. RA reduced HDMEC proliferation and differentiation, induced HDMEC apoptosis as well, as least in part, via over-expression of TSP-1 in vitro. |
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